Abstract

OBJECTIVE Increased oxidative stress (OS) and impaired anti-OS defenses are important in the development and persistence of insulin
resistance (IR). Several anti-inflammatory and cell-protective mechanisms, including advanced glycation end product (AGE)
receptor-1 (AGER1) and sirtuin (silent mating-type information regulation 2 homolog) 1 (SIRT1) are suppressed in diabetes.
Because basal OS in type 2 diabetic patients is influenced by the consumption of AGEs, we examined whether AGE consumption
also affects IR and whether AGER1 and SIRT1 are involved.

Insulin action is regulated by multiple factors, including SIRT1, a member of the sirtuin (silent mating type information
regulation 2 homolog) 1 family of NAD+ deacetylases, which acts via signaling mediators and transcription factors, including nuclear factor-κB (NF-κB), forkhead
box class O, peroxisome proliferator–activated receptor-γ, and adiponectin (1–3). Because SIRT1 activity also modulates the functions of monocytes and macrophages, native defenses may play a key role in
insulin resistance (IR), and type 2 diabetes (3,4). SIRT1 activity is decreased in diabetes (4,5), as is advanced glycation end product (AGE) receptor-1 (AGER1) (6). Because AGEs are oxidants that are normally controlled by AGER1 (7–9), decreased AGER1 may result in increased oxidative stress (OS) and inflammation (6). In addition, exposure to glycoxidants when AGER1 levels are reduced may have a negative effect on SIRT1, which could contribute
to IR.

The increasing prevalence of IR and type 2 diabetes is directly related to the Western lifestyle and diet (10). Excessive intake of fats or carbohydrates is thought to play a major role in the development of IR (11,12), although a direct link has not been established. A clear relationship has been found between inflammation, OS, and IR (13). Therefore, because AGEs increase inflammation and OS in normal subjects, as well as in diabetic patients, they may also
play a role in IR.

Glycoxidants are partially absorbed as food-derived AGE peptides and AGE lipids by mechanisms not fully elucidated (14). Experiments in animals have shown a strong link between high oral glycoxidant intake, IR, type 2 diabetes, and diabetes
complications (15–18). Direct evidence that oral AGEs promote OS and cause metabolic changes was provided by studies of mice that were pair fed
a low-AGE diet or the same diet supplemented with a well defined AGE (methylglyoxal [MG]-BSA) (16). An excess of AGEs led to OS, IR, and renal/vascular disease, whereas restriction of AGEs, without altering caloric or nutrient
intake, reduced OS and inflammation, ameliorated IR, and extended the life span in mice (16). Clinical studies in healthy subjects, diabetic patients, and individuals with chronic kidney disease showed that AGE restriction
substantially reduced OS and inflammation and improved native defenses, including AGER1 (9,19).

In the current study, we investigated the relationship between IR and dietary AGEs in type 2 diabetic patients. We report
that AGE restriction lowers insulin levels, markers of IR, and inflammation. In addition, the suppressed expression and function
of AGER1 and SIRT1 in diabetic peripheral blood mononuclear cells (PMNCs) are nearly normalized by AGE restriction, consistent
with restoration of host defenses.

Baseline clinical characteristics and other parameters in the study population

They were randomly assigned to an AGE-restricted diet (n = 12) or their usual diet (n = 6) for 4 months. The AGE-restricted group received instructions on how to modify cooking time and temperature but not the
quantity or nutrient composition of food. They were advised to boil, poach, stew, or steam food and to avoid frying, baking,
or grilling, methods, which limit dietary AGE intake by ∼40–50% (9,19,20). The control group comprised 18 age-matched (age 67 ± 1.4 years), healthy volunteers selected from a larger well characterized
cohort that was randomized to the same AGE-restricted diet for 4 months (9).

A dietitian monitored participants by telephone 1–2 times per week and at monthly clinic visits. The study diets were designed
to maintain daily requirements of calories and nutrients in both arms but to restrict AGE intake only on the AGE-restricted
arm. Nutritional intake was closely monitored in both groups.

The study and consent forms were approved by the Mount Sinai School of Medicine Institutional Review Board. All participants
provided informed consent.

Human subjects

Participants received a physical examination and provided a fasting blood sample and a 24-h urine collection at baseline and
at 4 months. Routine blood and urine tests were performed by the hospital clinical laboratory.

Dietary intake

Daily dietary AGE content, from 3-day food records that emphasized cooking methods, was estimated from a database that listed
AGE values of ∼560 foods and was expressed as AGE equivalents/day (AGE Eq = 1,000 kilounits) (20). The 3-day food record is based on established guidelines developed to allow estimation of food and beverage consumption
at home and away from home. The dietitian reviewed the record with the subject, probing for details on portion sizes and preparation
methods, to ensure accuracy and compliance. Nutrient calculations were estimated by using the Food Processor 10.1 nutrient
software program (ESHA Research, Salem, OR).

PMNCs

PMNCs were separated from fasting, EDTA-anticoagulated blood by Ficoll-Hypaque Plus gradient (American Biosciences, Uppsala,
Sweden) and used to isolate mRNA and protein (9,19). Total RNA was extracted by TRIzol (Invitrogen-Molecular Probes, Inc., Eugene, OR). The extracted RNA had an optical density
of 280/260 ratio of between 1.8 and 2.0. Total RNA was reverse-transcribed using Superscript III RT (Invitrogen, Carlsbad,
CA).

Emission from each sample was recorded during thermal cycling, and the raw fluorescence data were processed using Sequence
Detection System software (Applied Biosystems, Carlsbad, CA) to produce threshold cycle (Ct) values for each sample. β-Actin
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genes were used for internal normalization. The transcript
copy number of target genes was determined based on their Ct values (9).

NF-κB p65 acetylation

Cells (PMNCs, THP-1) were disrupted in lysis buffer (Sigma-Aldrich, C2978), and 100 µg protein was immunoprecipitated with
anti-NF-κB-p65 (Santa Cruz Biotechnology) at 4°C overnight. A total of 60 µL protein agarose (A/G) beads were added, and immunoprecipitates
were immunoblotted for acetyl-lysine residues (4). For densitometric analysis, acetyl-p65 was normalized for total p65.

NAD/NADH determination

NAD and NADH were measured using an Amplite Fluorimetric NAD/NADH assay kit (AAT Bioquest, Inc., Sunnyvale, CA). Cells (wild-type
or AGER1-transfected) were lysed after stimulation with MG-BSA (60 µg/mL) for 72 h in the presence or absence of antioxidants
NAC (5 mmol/L) and apocynin (300 µmol/L).

Statistical analysis

Data in the tables and figures are presented as means ± SEM. The Kolmogorov-Smirnov goodness-of-fit test was used to test
for normal distribution. Variables not normally distributed were logarithmically converted for analyses. Differences of means
between groups were analyzed by the Student t test or ANOVA, followed by the Bonferroni correction for multiple comparisons, depending on the number of groups. Correlation
analyses were also examined by the Pearson correlation coefficient.

Significance of changes during the interventional study was assessed by comparing 1) change of means between baseline and end of study within each group by paired t test, 2) percentage of change from baseline to the end of the study between the AGE-restricted and the regular AGE diet groups by
the Mann-Whitney test, and 3) differences between the means of both groups at the end of the study by unpaired t test. Significant differences were defined as a value of P < 0.05 and are based on two-sided tests. Data analysis was performed in consultation with a statistician, using SPSS 17.0
software (SPSS, Chicago, IL).

AGER1 and SIRT1 mRNA levels in PMNCs from diabetic subjects were significantly lower than in healthy subjects (Table 1). Concentrations of pro-OS p66shc and NAPDH oxidase p47phox mRNA and TNF-α protein, a proinflammatory cytokine, were elevated, as were intracellular AGEs (iCML; Table 1). These data were consistent with high baseline OS.

Intervention data

Serum and plasma changes after AGE restriction.

AGE restriction (by 50%), without altering nutrient intake, led to markedly lower levels of sCML and sMG, as well as 8-isoprostanes
in type 2 diabetic subjects (Table 2, Fig. 1A). Plasma insulin, HOMA, and leptin levels were also decreased by ∼30% below baseline by AGE restriction (Table 2, Fig. 1A). However, adiponectin levels were doubled after AGE restriction, resulting in a markedly lower leptin/adiponectin ratio
(Fig. 1A, inset).

A: AGE restriction reduces IR and improves inflammation in type 2 diabetic patients. Changes after AGE restriction (×4 months)
in circulating factors (by ELISAs), plasma insulin, and HOMA, leptin, adiponectin, serum CML, MG, or in PMNCs, and AGER1,
SIRT1, RAGE mRNA (by RT-PCR), are shown as percentage (mean ± SEM) above or below the baseline. *P < 0.050. Inset: Leptin (Lep)/adiponectin (Adipo) ratio before and after treatment is shown relative to normal control subjects (NL, at baseline,
open bars). B: AGE restriction enhances SIRT1 and AGER1 protein expression in PMNCs of type 2 diabetic patients. PMNCs obtained at entry
(1) and at the end of the study (2) are shown for subjects exposed to AGE restriction (AGE-Restr) vs. regular diet (Reg). SIRT1 and AGER1 protein levels were
assessed by Western blotting (upper panels), followed by densitometric analysis (lower panels). C: AGE restriction enhances SIRT1 deacetylation of NF-κB p65. Levels of NF-κB p65 acetylation are shown at entry (1) and at the end of study (2) after immunoprecipitation (IP) and immunoblotting (IB) against acetyl-lysine residues. Data are shown as the percentage
(mean ± SEM) change from entry (1). P values are as indicated.

PMNC changes in the AGE-restriction cohort.

The suppressed basal mRNA levels of AGER1 and SIRT1 in type 2 diabetic patients were increased by ∼50%, almost to the normal
range in the AGE-restricted cohort (Fig. 1A; Table 2) (9). The changes in mRNA of AGER1 and SIRT1 correlated with changes in protein expression (Fig. 1B). Intracellular AGE levels in the AGE-restricted group were decreased, iMG (P = 0.002) and iCML (P = 0.148; Table 2), consistent with increased AGER1 expression.

There was also a reduction in NF-κB acetyl-p65 (Fig. 1C), associated with trends of lower RAGE mRNA and TNF-α protein in PMNCs from AGE-restricted type 2 diabetic subjects (Table 2). In contrast, in diabetic subjects who ate the regular diet, PMNC SIRT1 remained depressed (Fig. 1B and C; Table 2), whereas acetyl-p65, TNF-α, and RAGE remained elevated in diabetic subjects, consistent with higher circulating oxidants,
AGEs, and 8-isoprostane (Table 2).

In vitro effects of AGEs on THP-1 AGER1 and SIRT1 expression and function

Because SIRT1 (4,23) and AGER1 (6–8) are suppressed in patients with diabetes (high OS), monocyte-like THP-1 cells were used as inflammatory cells to investigate
the observations in PMNCs. AGER1 and SIRT1 protein expression levels were coordinately suppressed in THP-1 cells after chronic
exposure to CML or MG (but not unmodified BSA; Fig. 2A). In addition, AGER1 and SIRT1 expression was enhanced in AGER1-overexpressing (AGER1+) cells treated with MG but reduced in AGER1-silenced THP-1 cells (Fig. 2B and C). After a transient induction, a marked time–dependent reduction of the NAD+/NADH ratio was noted in association with suppressed SIRT1 levels, and this effect was prevented by antioxidants (NAC, apocynin;
Fig. 2D) and by AGER1 overexpression (AGER1+; Fig. 2E). In addition, the AGE-induced NF-κB p65 hyperacetylation seen in wild-type cells was blocked in AGER1+ cells (Fig. 2F), consistent with preserved SIRT1 function when AGER1 is intact. These data were in line with the PMNC findings from type
2 diabetic patients.

CONCLUSIONS

IR remains an unresolved scientific and therapeutic problem. Increased OS and impaired anti-OS defenses are important factors
in the development and persistence of IR (2–4). The current study shows that AGE restriction can improve IR in type 2 diabetes. Plasma insulin, markers of inflammation,
and OS were substantially improved in diabetic subjects by an intervention that restricts oxidant (AGE) load without altering
energy balance. These findings are consistent with evidence showing that excessive food AGEs promote inflammation and OS in
humans (9,19) and IR in mice (15,16). That plasma insulin was nearly normalized after the AGE restriction indicates that tissue glucose uptake was improved.
Therefore, a reduced demand on β-cells for insulin release appears to be directly related to the AGE restriction. While the
findings must be confirmed in larger cohorts, AGE restriction may have therapeutic advantages in IR.

Although endogenous glucose or lipids contribute to elevated AGEs (22), exogenously AGE-modified nutrients are an important source in both human subjects and animals (9,14,16,19,20). The significant reduction in serum AGEs and lipid peroxides (8-isoprostanes) after AGE restriction in the current study
confirms the hypothesis that elevated AGEs in type 2 diabetes result from increased dietary AGE intake, independently from
glycemic control (19).

AGE restriction normalized AGER1, SIRT1, and SIRT1-dependent NF-κB p65 deacetylation levels, and there was a concomitant attenuation
of proinflammatory mediators (i.e., RAGE and TNF-α) (9). The decreased basal AGER1 and SIRT1 (mRNA and protein) levels in type 2 diabetic PMNCs suggests a link between these two
mechanisms. Since plasma insulin and leptin levels fell, whereas adiponectin levels increased with AGE restriction, the changes
in AGER1 expression reflected adjustments in both the inflammatory and the metabolic states, effects previously attributed
to SIRT1 (2–4,23). Thus, high AGE and low AGER1 levels in type 2 diabetes may partly account for the persistence of IR in these patients.

Given that downregulation of AGER1 (7–9) and SIRT1 is linked to high OS conditions (4,5), the current findings also indicate that normal SIRT1 function may depend on the ability of intact AGER1 to control AGEs
and OS. Thus, the loss of AGER1 function may be one mechanism leading to SIRT1 downregulation in type 2 diabetes. This postulate
is supported in the current study by the high basal levels of intracellular AGEs and low AGER1 levels in type 2 diabetic PMNC.
The fact that these levels are nearly normalized after AGE restriction lends further support to this hypothesis. These data
also suggest that low steady-state levels of intracellular AGEs may be crucial for maintaining AGER1 intact (7–9,16,24).

Because this was the first time that AGEs and AGER1 were linked to the deacetylases and to SIRT1, we probed these relationships
in inflammatory monocyte–like THP-1 cells. After prolonged exposure to AGEs, AGER1 and SIRT1 were both suppressed in these
cells or became unresponsive. AGEs acted via an OS-dependent decrease in NAD+, explaining the eventual downregulation of SIRT1, and consequently, the hyperacetylation of NF-κB p65 observed in diabetic
PMNCs (2,4). Since the AGE-induced effects on SIRT1 and NF-κB were blocked in THP-1 cells by genetic modulation of AGER1 expression,
AGER1 is likely to be involved in the regulation of inflammatory responses controlled by SIRT1. The data also suggest that
controlling the pro-oxidant effects of AGEs could help preserve normal AGER1 and SIRT1 functions.

From a clinical perspective, AGE restriction markedly increased adiponectin levels and reduced the initially high leptin/adiponectin
ratio, a marker of IR, indicating that AGEs also affect insulin-sensitive tissues (2–5). Adiponectin improves insulin sensitivity and is controlled by SIRT1 (25); therefore, the doubling in adiponectin levels after AGE-restriction provides another insight into the improved metabolic
state in type 2 diabetic patients.

These data show that chronic exposure to excessive exogenous (food) oxidants fosters an impairment in both native antioxidant
defenses as well as insulin action and that reducing oral AGEs can ameliorate these defects. This approach does not restrict
energy or caloric intake. Since the effects of AGE restriction were additive to standard medical therapy, the intervention
could be a valuable addition to the current management of patients with type 2 diabetes. Larger trials are warranted to validate
the findings and establish the long-term effects of this intervention on diabetes.

Acknowledgments

This work was supported by MERIT grants AG-23188 and AG-09453 (to H.V.) and from the National Institutes of Health, National
Institute of Research Resources (Grant M01-RR-00071) to the General Clinical Research Center at Mount Sinai School of Medicine
for clinical and statistical support.

No potential conflicts of interest relevant to this article were reported.

J.U. researched the data and reviewed and edited the manuscript. W.C. researched the data and wrote, reviewed, and edited
the manuscript. M.R. researched the data. S.G. researched the data and contributed to the discussion. R.P., X.C., and L.Z.
researched the data. G.E.S. reviewed and edited the manuscript. H.V. designed the study, obtained support, researched the
data, contributed to the discussion, and wrote, reviewed, and edited the manuscript.

Part of this study was submitted in abstract form to the 71st Scientific Sessions of the American Diabetes Association, 24–28
June 2011, San Diego, California.

. Protection against loss of innate defenses in adulthood by low advanced glycation end products (AGE) intake: role of the antiinflammatory
AGE receptor-1. J Clin Endocrinol Metab2009;94:4483–4491pmid:19820033